Polymorphonuclear granulocytes (PMNs) are critical elements in human host defense. The most efficient PMN microbicidal system depends on two elements: reactive oxygen species generated by activation of the NADPH- dependent oxidase and myeloperoxidase (MPO), a heme-containing lysosomal protein present exclusively in cells of PMN and monocyte lineage. In our studies of MPO biosynthesis we noted two peculiar features which serve as stimuli for this proposal. First, processing of MPO precursor to mature MPO is extremely slow, suggesting that biosynthetic intermediates in the endoplasmic reticulum (ER) must avoid aggregation and retain their capacity for function for a relatively long time. Second, the processing and proteolytic maturation of MPO precursor to native lysosomal MPO requires insertion of heme into the precursor. Inhibition of heme synthesis results in a maturation arrest in MPO processing. For these reasons we speculated that: (1) heme insertion may be a rate-limiting step in the biosynthesis of MPO and (2) molecular chaperones may interact with MPO precursors and facilitate folding events necessary for insertion of heme into heme-free MPO precursor(s). In this proposal we outline studies to test the general hypothesis that specific ER-resident proteins are critical participants in the biosynthesis of myeloid heme proteins. We present data that calreticulin (CRT) and calnexin (CLN), two calcium-binding ER-resident proteins, specifically interact with precursors of MPO during its biosynthesis. Furthermore, we show that CRT interacts exclusively with apoproMPO, the heme-free precursor, whereas CLN interacts with apoproMPO and with a subset of proMPO molecules. We propose studies to characterize the nature of the interactions of MPO precursors with CRT and CLN, to define the function of these interactions, and to explore the full range of myeloid proteins served by CRT and CLN as molecular chaperones. To achieve these ends we have identified three specific aims: 1) to characterize in depth the interactions of CRT and CLN with MPO precursors; 2) to determine if CRT and/or CLN participate in heme incorporation into apoproMPO; and 3) to determine what peptide motifs mediate binding to CRT/CLN in their capacity as molecular Chaperones. We anticipate these studies will provide new information regarding the role of molecular chaperones in myeloid protein synthesis. In addition, insights into these specialized early folding events may be applicable to events in the biosynthesis of other myeloid hemeproteins and animal peroxidases in general.